Rosa Freitas

Benthic biotopes remote sensing using acoustics

Abstract The present work applies novel methodologies to the study of sublittoral benthic biotopes, by combining the information given by underwater acoustic and biological survey techniques. The acoustic seabed classification system QTC VIEW™ was used to map the acoustic diversity between 5 and 40 m water depth on the shelf off “Ria de Aveiro”, Western coast of Portugal. Ground-truth was undertaken using an analysis of superficial sediments grain-size, and compared to the species composition and distribution of macrofaunal communities. Sedimentary and biological data were submitted to ordination analysis, and the acoustic data to both ordination and cluster analysis. The acoustic classes identified were mapped using a geographical information system. The acoustic results showed a very clear geographic pattern, with the acoustic classification being coincident where survey lines crossed, confirming the stability of the classification procedure. At the optimal splitting level, three acoustic classes were obtained. These classes were correlated to differences in coarse, fine and very fine sands. Additional real bottom differences in the grain-size of the coarser sand classes were not detected by the acoustic method, possibly due to the fact that they showed a similar degree of compactness. The benthic ecological data suggests only two main communities, which correspond to the outcomes of a two-class acoustic split. Therefore, a mismatch was noticed between the optimal acoustic split and the number of major biological communities present. However, by dropping the acoustic solution down to two classes, an optimal relationship to benthic communities is achieved. Overall, results suggest that the acoustic system provides very valuable and important data for mapping soft sediment biotopes, even in areas of relative bottom monotony such as the one analysed, but careful ground-truth is required to ensure that the acoustic class splits are biologically relevant.

Presence of the pharmaceutical drug carbamazepine in coastal systems: effects on bivalves.

Carbamazepine (CBZ), an antiepileptic drug, is one of the most commonly detected pharmaceutical drugs in aquatic ecosystems, and is used as a marker of urban pollution. Since CBZ is designed to exert a biological effect, when it reaches aquatic environment high probability exist for toxic effects on non-target organisms. The present study evaluated the acute toxicity of environmentally relevant concentrations of CBZ (0.00, 0.03, 0.30, 3.00, 9.00μg/L) in the edible clams Venerupis decussata (a native species) and Venerupis philippinarum (an invasive species) collected from the Ria de Aveiro. The effects on both species were assessed through the use of a battery of biomarkers mainly related with health status and oxidative stress. Furthermore, in this work an alternative and promising tool, the direct competitive immunoassay ELISA, for the direct CBZ quantification in clams tissues, was applied. The results of the present work showed that CBZ in clams tissues increased with the exposure concentration and V. decussata gave slightly higher values than V. philippinarum. Although the clams accumulated lower levels of CBZ than the concentration of exposure, these concentrations were enough to impair the health status and induce oxidative stress. However, a different response to CBZ was observed in the two species. While in V. philippinarum the lipid peroxidation levels increased at the highest CBZ concentration (9.00μg/L), in V. decussata a significant decrease was seen. Moreover, glutathionse S-transferase activity was stimulated in V. decussata and decreased in V. philippinarum. Nevertheless, an induction of glutathione reductase, superoxide dismutase and cytochrome P450 3A4 activities was found in both species as a result of the exposure. The results indicate that, probably, V. philippinarum have a less efficient antioxidant system than V. decussata, and are therefore less capable to neutralize oxidative stress and consequently more sensitive to CBZ. The risk quotient determined for the Ria de Aveiro was higher than 1 indicating that a ecotoxicological risk is suspected. Furthermore, bioaccumulation of CBZ in clams should be taken into consideration since this chemical might be transferred along the food chain and affect non-target organisms.

Looking for suitable biomarkers in benthic macroinvertebrates inhabiting coastal areas with low metal contamination: Comparison between the bivalve Cerastoderma edule and the Polychaete Diopatra neapolitana

Metals accumulated in marine sediments are often a threat to benthic communities. With the recognized importance and wide use of stress biochemical responses as indicators of metal contamination it becomes essential to compare these markers between different species and verify their ubiquity and accuracy. Using wild Diopatra neapolitana and Cerastoderma edule, collected at several areas differing in metal contamination, this study aimed to assess the use of these two macrobenthic species as sentinel organisms and to determine the applicability of currently used biomarkers in benthic species exposed to a range of low metal and As concentrations. Total metal accumulation and intracellular partitioning was analyzed and metal-induced alterations were assessed through the analysis of several biochemical parameters in both organisms, including stress-induced reactive oxygen species (ROS), lipid peroxidation and protein content, the activity of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and of glutathione S-transferase (GST). Metal chelation by Metallothioneins (MTs) was also determined. Results revealed that D. neapolitana accumulated higher levels of metals when compared to C. edule, independently of the sediment concentration. Results also showed strong species-specific responses to metals and differences in the ability to sequester metals. Overall, C. edule showed to be more efficient metal chelator and precipitatior than D. neapolitana, which was less tolerant and presented oxidative stress. MTs proved to be a good predictor of metal accumulation in both species, even under low metal exposures. On the other hand, lipid peroxidation was a good indicator of oxidative damage, only observed in D. neapolitana, which was a result of higher metal retention in the soluble fraction.

Biochemical effects of acetaminophen in aquatic species: edible clams Venerupis decussata and Venerupis philippinarum

Acetaminophen (paracetamol) is one of the most used pharmaceutical drugs, due to its antipyretic and analgesic properties that turn it into a primary choice in varied pathologies and conditions. However, and despite its massive use, acetaminophen is not exempt of adverse effects, especially when administered in over dosage, which are related to the formation of toxic metabolites by oxidative pathways. It is thus possible to observe that toxicity caused by acetaminophen is usually mediated by reactive oxygen species and can result in multiple effects, ranging from protein denaturation to lipid peroxidation and DNA damage. The occurrence of acetaminophen has been reported in the aquatic environment, being important to address the potential exertion of toxic effects on nontarget environmentally exposed organisms. The present study intended to characterize the effects of acute acetaminophen exposure on physiological traits (antioxidant defense, oxidative damage) of two species of bivalves, namely, the edible clams Venerupis decussata and Venerupis philippinarum. Results showed a significant increase in all oxidative stress biomarkers, evidencing the bioactivation of acetaminophen into a deleterious prooxidant, triggering the onset of deleterious effects. Furthermore, strong interspecific differences were observed among responses of the two tested species, which was a major issue due to intrinsic ecological implications when one considers that both species share the same habitat.

Acoustic seabed classification of marine habitats: studies in the western coastal-shelf area of Portugal

Two single-beam, seabed-classification systems, QTC VIEW Series IV and QTC VIEW Series V, were used to identify and map biosedimentary gradients in a mid-shelf area off Western Portugal. The survey area has a moderate slope, a depth ranging from 30 to 90 m along a 3.5-km axis perpendicular to the shoreline, and is characterized by smooth sedimentary and biological gradients. Ground truth for sediment grain size and macrofaunal communities was based on grab sampling at 20 sites. The sedimentary and biological data were analysed using classification and ordination techniques. The acoustic data were analysed with qtc impact software and classified into acoustic classes. The affinity groups obtained in each data set were mapped using a Geographics Information System. All showed good agreement and identified prevailing gradients along a northwest-southeast direction. Three acoustic classes were identified, corresponding to the predominant sediment types, namely fine sand with low silt and clay content, silty, very fine sand, and mud. A close relationship with benthic communities was also verified, although less marked because benthic communities continuously change along the northwest-southeast gradient. Overall, the acoustic system coupled with ground-truthing data was able to discriminate and characterize the various benthic biotopes in the survey area.

Physiological and biochemical responses of three Veneridae clams exposed to salinity changes.

Given their global importance, coastal marine environments are a major focus of concern regarding the potential impacts of climate change, namely due to alterations in seawater salinity. It is known that environmental characteristics, such as salinity, affect immune and physiological parameters of bivalves. Nevertheless, scarce information is available concerning the biochemical alterations associated with salinity changes. For this reason, the present work aimed to evaluate the biochemical responses of three venerid clam species (Venerupis decussata, Venerupis corrugata, Venerupis philippinarum) submitted to salinity changes. The effects on the native (V. decussata and V. corrugata) and invasive (V. philippinarum) species collected from the same sampling site and submitted to the same salinity gradient (0 to 42g/L) were compared. The results obtained demonstrated that V. corrugata is the most sensitive species to salinity changes and V. decussata is the species that can tolerate a wider range of salinities. Furthermore, our work showed that clams under salinity associated stress can alter their biochemical mechanisms, such as increasing their antioxidant defenses, to cope with the higher oxidative stress resulting from hypo and hypersaline conditions. Among the physiological and biochemical parameters analyzed (glycogen and protein content; lipid peroxidation levels, antioxidant enzymes activity; total, reduced and oxidized glutathione) Catalase (CAT) and especially superoxide dismutase (SOD) showed to be useful biomarkers to assess salinity impacts in clams.

Physiological and biochemical responses of the Polychaete Diopatra neapolitana to organic matter enrichment

Several studies have demonstrated that organic matter enrichment may be associated to aquaculture, leading to impoverished benthic communities and species succession with loss of biodiversity, but very few studies have investigated biochemical and physiological alterations that species affected by aquaculture activities undergo. Thus, in the present study, the effects of the organic enrichment originating from an oyster culture were studied in the Polychaete Diopatra neapolitana, a species already shown to be sensitive to inorganic contamination. For this, physiological responses and biochemical alterations were evaluated. The results obtained revealed that individuals from highly organically enriched areas presented lower capacity to regenerate their body but higher glycogen and protein levels. Furthermore, with increasing organic matter D. neapolitana increased the lipid peroxidation (LPO), the oxidized glutathione content (GSSG) and Glutathione S-transferase activity (GSTs) content, and the activity of the antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD). This study evidenced that organic matter enrichment induced biochemical and physiological alterations in D. neapolitana. Thus, this species was shown to be a good sentinel species to monitor organic contamination.

The effects of carbamazepine on macroinvertebrate species: Comparing bivalves and polychaetes biochemical responses.

In the present study, the bivalve Scrobicularia plana and the polychaete Diopatra neapolitana were exposed to an increasing carbamazepine (CBZ) concentration gradient. Both species are among the most widely used bioindicators, and CBZ is one of the most commonly found drugs in the aquatic environment. After a chronic exposure (28 days), the results obtained revealed that CBZ induced biochemical alterations in both species. Our findings demonstrated that S. plana and D. neapolitana reduced the CBZ accumulation rate at higher CBZ concentrations, probably due to their capacity to decrease their feeding rates at stressful conditions. Nevertheless, this defence mechanism was not enough to prevent both species from oxidative stress. In fact, S. plana and D. neapolitana were not able to efficiently activate their antioxidant defence mechanisms which resulted in the increase of lipid peroxidation, especially at the highest CBZ concentrations. Comparing both species, it seems that S. plana was the most sensitive species since stronger biochemical alterations were observed in this species.

Ruditapes decussatus and Ruditapes philippinarum exposed to cadmium: toxicological effects and bioaccumulation patterns.

Since differences in metal accumulation may exist between bivalve species, the aim of this study was to assess the impact of cadmium (Cd) on Ruditapes decussatus and Ruditapes philippinarum. For this, the Cd accumulation, mortality rates and biochemical responses were analysed in the two species after 5days of exposure, under laboratory-controlled conditions. The concentration of Cd that caused 50% of mortality on clams was two-times higher in R. decussatus than in R. philippinarum. For both species, higher percentage (84.5-98.2%) of the Cd was in the insoluble fraction, but the Cd concentration in solution was 3 to 8 times higher in R. decussatus. Nevertheless, R. philippinarum presented higher oxidative stress and higher CAT activity. The paradox observed between the two clams can be explained by the higher capacity of R. decussatus to increase the expression of MTs when exposed to Cd.

The effects of arsenic and seawater acidification on antioxidant and biomineralization responses in two closely related Crassostrea species.

Ocean acidification processes are major threats to marine calcifying organisms, mostly affecting biomineralization related processes. Abiotic stressors acting on marine systems do not act alone, rather in a combination of multiple stressors, especially in coastal habitats such as estuaries, where anthropogenic and environmental pressures are high. Arsenic (As) is a widely distributed contaminant worldwide and its toxicity has been studied on a variety of organisms. However, the effect of low pH on the toxicity of As on marine organisms is unknown. Here, we studied the combined effects of ocean acidification and As exposure on two closely related oyster species (Crassostrea angulata and Crassostrea gigas), by use of a biochemical approach. Oxidative stress related parameters were studied along with the assessment of biomineralization enzymes activity after 28days of exposure. Results showed that both species were sensitive to all tested conditions (low pH, As and pH+As), showing enhancement of antioxidant and biotransformation defenses and impairment of biomineralization processes. Glutathione S-transferases (GSTs) activity were significantly higher in oysters exposed to As, showing activation of detoxification mechanisms, and a lower GSTs activity was observed in low pH+As condition, indicating an impact on the oysters capacity to detoxify As in a low pH scenario. Carbonic anhydrase (CA) activity was significantly lower in all tested conditions, showing to be affected by both As and low pH, whereas the combined effect of low pH+As was not different from the effect of low pH alone. Multivariate analysis of biochemical data allowed for the comparison of both species performance, showing a clear distinction of response in both species. C. gigas presented overall higher enzymatic activity (GSTs; superoxide dismutase; catalase; CA and acid phosphatase) and higher cytosolic GSH content in As exposed oysters than C. angulata. Results obtained indicate a higher tolerance capacity of the Pacific oyster C. gigas towards the tested conditions.